ABSTRACT Alcohol is the most common addictive substance in the world. Alcohol-use disorders, which affect 17 million people in the United States alone, represent a major health issue and a considerable cost to society. One of the major challenges in the alcohol research field is to understand the neurobiological bases of the progressive transition from low/moderate social drinking to the heavy alcohol use which characterizes alcohol dependence. Repeated intoxications and withdrawal produce profound neuroadaptations in specific brain regions critical for the reinforcing effects of alcohol, such as the extended amygdala. In particular, chronic alcohol exposure causes neuroadaptations in the bed nucleus of the stria terminalis (BNST), a brain area which plays a key role in chronic, pathological ethanol use and in withdrawal-induced negative emotional states. The BNST is very rich in a neuropeptide called pituitary adenylate cyclase-activating polypeptide (PACAP), which is considered a master regulator of the major stress circuits in the brain. We have observed that excessive intermittent alcohol consumption causes a marked increase in PACAP levels in the BNST of both male and female mice, compared to control mice, suggesting that PACAP in the BNST may play a key role in regulating high levels of alcohol drinking. The central hypothesis of this application is that PACAP neurons of the BNST represent a major system responsible for the transition to heavy alcohol use and alcohol dependence. We will test these hypotheses by means of a combined behavioral, neuroanatomical, molecular, and chemogenetic approach, which involves the use of PACAP-Ires-Cre mice. Specific Aim 1 will further validate the PACAP-Ires-Cre mouse line, characterize BNST PACAP neurons by examining its co-expression with other neuropeptide markers abundant in this brain region, and characterize BNST PACAP projection targets by means of an adeno-associated virus (AAV) expressing a Cre-dependent fluorescent reporter gene. Specific Aim 2 will then determine the functional role of PACAP neurons of the BNST in the transition to alcohol dependence and heavy alcohol drinking, using a chemogenetic approach. If successful, these experiments will lay the foundation for more extensive studies which may lead to the identification of a neuropeptide system with a critical role in heavy alcohol drinking and in the transition to dependence. A deeper understanding of the neuroadaptations produced by chronic alcohol will be essential for the discovery of novel therapeutic agents to alleviate alcoholism.